Composition and method for treating neurodegenerative disease

ABSTRACT

The present invention provides a method and composition for treating a neurodegenerative disease. In particular, the present invention provides a method and composition to increase DJ-1 gene expression or DJ-1 protein activity to treat a neurodegenerative disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/273,404, filed Sep. 22, 2016, now allowed, which is a continuation ofU.S. patent application Ser. No. 14/388,929, filed Sep. 29, 2014, nowU.S. Pat. No. 9,480,673, which is a 35 U.S.C. § 371 national stageapplication of, and claims priority to, International Patent ApplicationNo. PCT/US13/34501, filed Mar. 28, 2013, which claims priority under 35U.S.C. § 119(e) to U.S. Provisional Patent Application No. 61/617,055,filed Mar. 29, 2012, all of which applications are incorporated hereinby reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for treating aneurodegenerative disease. In particular, the present invention relatesto increasing DJ-1 gene expression or DJ-1 protein activity to treat aneurodegenerative disease.

BACKGROUND OF THE INVENTION

Neurodegenerative diseases such as Parkinson's disease, Alzheimer'sdisease and Huntington's disease are caused by the death of neurons. Asused herein, the term “neurodegenerative” refers to the loss or death ofneurons causing the disease. Typically, neurodegenerative diseases arecharacterized by a slow onset and chronic progression. In aneurodegenerative disease, a particular part of the brain, spinal cord,or peripheral nerve functionally fails and the neurons of thedysfunctional region die. Neurodegenerative diseases are oftencategorized by whether they initially affect cognition, movement,strength, coordination, sensation, or autonomic control. However, it isnot uncommon for patients to be presented with symptoms and signs tomore than one system. While it is possible that involvement of severalsystems can occur concomitantly, typically by the time the patient hasfunctionally declined enough to seek medical attention multiple systemshave become involved. Diagnosing neurodegenerative diseases can proveparticularly intimidating to clinicians, because many times thediagnosis cannot be critically “confirmed” by a simple test.

The prevalence of neurodegenerative diseases has increased drastically.In fact, it is estimated that currently Alzheimer's disease is now thesixth leading cause of death in the U.S.

Accordingly, there is an urgent need for effective treatments ofneurodegenerative diseases.

SUMMARY OF THE INVENTION

Some aspects of the invention are based on the discovery by the presentinventors that increasing DJ-1 gene expression or increasing DJ-1protein activity can be beneficial to neurons. In particular, it hasbeen discovered by the present inventors that increasing DJ-1 geneexpression or DJ-1 protein activity protects dopamine producing neuronsfrom apoptosis. Accordingly, in some aspects of the invention provides amethod for treating a neurodegenerative disease in a subject, suchmethod typically comprising administering to a subject in need of such atreatment a composition comprising a compound that increases DJ-1 geneexpression or DJ-1 protein activity. The term “treating” or “treatment”of a disease includes: (1) preventing the disease, i.e., causing theclinical symptoms of the disease not to develop in a mammal that may beexposed to or predisposed to the disease but does not yet experience ordisplay symptoms of the disease; (2) inhibiting the disease, i.e.,arresting or reducing the development of the disease or its clinicalsymptoms; or (3) relieving the disease, i.e., causing regression of thedisease or its clinical symptoms.

In some embodiments, the compound increases DJ-1 gene expression by atleast 50%, typically at least 100%, often at least 200%, and most oftenat least 300%.

Yet in other embodiments, the compound is administered to the subject nomore than 10 units, typically no more than 5 units and often no morethan 3 units, per day.

Exemplary neurodegenerative diseases that can be treated by methodsand/or compositions of the invention include neurodegenerative diseasesassociated with a build-up of particular proteins such as, but notlimited to, α-synuclein or β-amyloid including amyloid angiopathy. Ingeneral, any neurodegenerative diseases that are associated withabnormal accumulation or build-up of a protein within or near theneurons can be treated by methods and compositions disclosed herein.Some specific examples of neurodegenerative diseases that can be treatedby methods and compositions of the invention include, but are notlimited to, Parkinson's disease, Alzheimer's disease, Huntington'sdisease, Multiple System Atrophy, Progressive Supranuclear Palsy, Downsyndrome, diffuse Lewy body disease, and amyotrophic lateral sclerosis.

Yet in other embodiments, the compound that can be used to treat aneurodegenerative disease is a histone deacetylase (HDAC) inhibitor.

In some embodiments, the compound that is used to treat aneurodegenerative disease upregulates DJ-1 gene expression. In otherembodiments, the compound increases the activity of DJ-1 protein.

In one particular embodiment, the compound comprises phenylbutyric acid,butytric acid, a salt thereof, a prodrug thereof, or a derivativethereof. In another embodiments, the compound comprises phenylbutyrateor a prodrug thereof, or a derivative thereof. Still in anotherembodiment, the compound comprises phenylbutyrate glycerol.

Yet another aspect of the invention provides a method for treating aneurodegenerative disease in a subject in need of such a treatment. Suchmethod generally includes increasing DJ-1 expression in the subject byadministering a therapeutically effective amount of a compound thatincreases DJ-1 expression level by at least 50%, typically at least100%, often at least 200%, and most often at least 300%. Increase inDJ-1 expression reduces the amount of neuron death. As used herein, theterm “a therapeutically effective amount” means the amount of a compoundthat, when administered to a mammal for treating a disease, issufficient to effect such treatment for the disease. It should beappreciated that the “therapeutically effective amount” will varydepending on the compound, the disease and its severity and the age,weight, etc., of the mammal to be treated.

Still another aspect of the invention provides a method for protectingdopamine neurons from a neurotoxic insult comprising administering acompound to increase DJ-1 expression in the dopamine neurons, therebyincreasing survivability of the dopamine neurons. It should beappreciated that whether a particular compound or a composition canincrease DJ-1 expression of DJ-1 protein activity can be readilydetermined by one skilled in the art having read the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 A is a western blot image showing sodium phenylbutyrate (PB) andsodium butyrate (SB) increases DJ-1 expression in N27 and HEK293 cells.

FIGS. 1B and 1C are quantitative data from Western blot images (*p<0.05,**p<0.01 compared to control, n=6) showing the amount of increase inDJ-1 expression in N27 and HEK293 cells by PB and SB.

FIGS. 1D and 1E are bar graphs showing the average luciferase activity(*p<0.05, **p<0.01 compared to control, n=6) in HEK293 reporter cellsexpressing human DJ-1 promoter-Luciferase that were treated with PB orSB at indicated doses for 48 hr.

FIGS. 2A-B are sample images of GFP adenovirus expression showingprotective effects of sodium phenylbutyrate in N27 cells that wereincubated with PB for 48 hr, followed by 24 hr treatment with H₂O₂ or6-OHDA at various concentrations.

FIGS. 2C and 2D are sample images of α-synuclein antibody LB509 stainingshowing protective effects of sodium phenylbutyrate in N27 cells thatwere incubated with PB for 48 hr, followed by 48 hr treatment ofadenovirus expressing A53T human α-synuclein (200 pfu/cell).

FIGS. 2E-2H are bar graphs of quantitative data from sample images of2A-2D, respectively. Three random fields (150-200 cells per field) wereexamined to determine the percentage of cells with α-synucleinaggregates (arrows in G-H). Triplicate treatments in 24-well plates wereused and experiments were repeated three times. (*p<0.05, **p<0.01compared to control, n=9). Cell viability was determined by MTT assays.

FIG. 3 A is a Western blot showing that knockdown of endogenous DJ-1abolishes neuroprotection from sodium phenylbutyrate.

FIGS. 3B and 3C are bar graphs showing cell viability data as measuredby MTT assay. (*p<0.05, **p<0.01 compared to control, n=9).

FIG. 4A is a Western blot of mouse brain tissues with DJ-1, α-synucleinand β-actin antibodies showing sodium phenylbutyrate and sodium butyrateincreased DJ-1 expression.

FIGS. 4B and 4C are quantitative data of DJ-1 and α-synuclein levelsafter PB and SB treatment (*p<0.05, **p<0.01 compared to control, n=4).

FIGS. 5A-5H are bar graphs (5A, 5C, 5D and 5E), Western blots (5B), andimages showing sodium phenylbutyrate prevented mouse dopamine neurondeath after MPTP lesion (Bar length, 1 mm for F-H).

FIGS. 6A-6D are graphs showing sodium phenylbutyrate improved motorfunction in aged α-synuclein transgenic mice (*p<0.05, **p<0.01, n=10).

FIGS. 7A-7D are graphs showing sodium phenylbutyrate improved cognitivefunction in aged α-synuclein transgenic mice (*p<0.05, **p<0.01, n=10).

FIGS. 8A-J are Western blots (8A and 8C), graphs of quantitative dataresults (8B and 8D), brain section images of Young and Old transgenicmice with or without PB treatment that were immunostained with humanα-synuclein antibody (LB509) (8E-8I), and a bar graph result showingthat sodium phenylbutyrate increased DJ-1 expression and reducedα-synuclein oligomer formation and aggregation in transgenic mice.

FIG. 9 is a graph showing transgenic animals treated with daily glycerolphenylbutyrate were able to stay on a rotating rod longer than animalsreceiving only glycerol.

FIG. 10 is a bar graph showing PB increases DJ-1 mRNA in N27 dopamineneurons.

FIG. 11 is a bar graph showing that the oxidative stress pathway Nrf2 isupregulated by phenylbutyrate.

FIG. 12 is a bar graph showing that the lysosomal pathway Lamp2 isactivated by phenylbutyrate in N27 dopamine neurons.

FIG. 13 is a bar graph showing that the vesicle-exosome protein Vamp2 inN27 dopamine neurons is upregulated by mutant protein expression withthe addition of phenylbutyrate.

DETAILED DESCRIPTION OF THE INVENTION

Parkinson's disease is caused by the death of midbrain dopamine neuronsdue to a variety of factors including, but not limited to, oxidativestress, abnormal protein aggregation, and genetic predisposition. In2003, Bonifati et al. found that a single amino acid mutation in theDJ-1 protein was associated with early-onset, autosomal recessiveParkinson's disease (PARK7). The mutation L166P prevents dimerizationthat is essential for the antioxidant and gene regulatory activity ofthe DJ-1 protein. Thus, the absence of normal DJ-1 protein causesParkinson's disease.

Some aspects of the invention are based on the discovery by the presentinventors that increasing the expression (i.e., overexpression) and/orthe activity of DJ-1 protein can be an effective treatment forneurodegenerative diseases such as Parkinson's disease. It should beappreciated that when referring to overexpressing or increasing theactivity of DJ-1 protein, the present disclosure refers tooverexpressing or increasing the activity of non-mutant DJ-1 protein.The term “mutant DJ-1 protein” refers to a DJ-1 protein having amutation that is associated with early-onset of neurodegenerativedisease, such as autosomal recessive Parkinson's disease. Thus, thecorollary term “normal DJ-1 protein” refers to any DJ-1 protein that isnot associated with an early-onset of neurodegenerative disease. As usedherein, the term “expression” refers to (1) the amount of transcriptionand/or translation of DJ-1 gene, (2) the amount of DJ-1 protein presentin the subject or (3) both. To detect expression of a gene refers to theact of actively determining whether a gene is expressed or not. This caninclude determining whether the gene expression is unregulated ascompared to untreated state. It should be appreciated that levels ofother biomarkers such as any portion of DJ-1 protein, DJ-1 mRNA, DJ-1gene expression, or ligand that can identify or correlate with the levelof DJ-1 protein can be used to determine the amount of DJ-1 proteinpresent in the subject.

Unless the context requires otherwise, the terms “baseline” or “control”refers to the level of DJ-1 protein expression in a subject withoutadministration or in the absence of any compound or composition thatincreases DJ-1 expression.

Expression of the transcripts and/or proteins encoded by DJ-1 gene canbe measured by any of a variety of known methods in the art. In general,the nucleic acid sequence of a nucleic acid molecule (e.g., DNA or RNA)in a patient sample can be detected by any suitable method or techniqueof measuring or detecting gene sequence or expression. Such methodsinclude, but are not limited to, polymerase chain reaction (PCR),reverse transcriptase-PCR (RT-PCR), in situ PCR, quantitative PCR(q-PCR), in situ hybridization, Southern blot, Northern blot, sequenceanalysis, microarray analysis, detection of a reporter gene, or otherDNA/RNA hybridization platforms. For RNA expression, typical methodsinclude, but are not limited to: extraction of cellular mRNA andNorthern blotting using labeled probes that hybridize to transcriptsencoding all or part of DJ-1 gene; amplification of mRNA expressed fromDJ-1 gene using gene-specific primers, polymerase chain reaction (PCR),quantitative PCR (q-PCR), and reverse transcriptase-polymerase chainreaction (RT-PCR), followed by quantitative detection of the product byany of a variety of means; extraction of total RNA from the cells, whichis then labeled and used to probe cDNAs or oligonucleotides encoding allor part of the genes of this invention, arrayed on any of a variety ofsurfaces; in situ hybridization; and detection of a reporter gene. Theterm “quantifying” or “quantitating” when used in the context ofquantifying transcription levels of DJ-1 gene can refer to absolute orto relative quantification. Absolute quantification may be accomplishedby inclusion of known concentration(s) of one or more target nucleicacids and referencing the hybridization intensity of unknowns with theknown target nucleic acids (e.g. through generation of a standardcurve). Alternatively, relative quantification can be accomplished bycomparison of hybridization signals to quantify the changes inhybridization intensity and, by implication, transcription level.

Methods to measure protein expression levels of DJ-1 gene include, butare not limited to, Western blot, immunoblot, enzyme-linkedimmunosorbant assay (ELISA), radioimmunoassay (RIA),immunoprecipitation, surface plasmon resonance, chemiluminescence,fluorescent polarization, phosphorescence, immunohistochemical analysis,matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)mass spectrometry, microcytometry, microarray, microscopy, fluorescenceactivated cell sorting (FACS), flow cytometry, and assays based on aproperty of DJ-1 including but not limited to ligand binding, orinteraction with other protein partners.

Nucleic acid arrays can also be used for detecting the expression ofDJ-1 gene. The production and application of arrays in gene expressionmonitoring have been disclosed previously in, for example, PCTPublication Nos. WO 97/10365, WO 92/10588, WO 95/35505, U.S. Pat. Nos.6,040,138 and 5,445,934, Hacia et al. (1996) Nature Genetics 14:441-447,Lockhart et al. (1996) Nature Biotechnol. 14:1675-1680, and De Risi etal. (1996) Nature Genetics 14:457-460, all of which are incorporatedherein by reference in their entirety. In general, in an array, anoligonucleotide, a cDNA, or genomic DNA, that is a portion of DJ-1 gene,occupies a known location on a substrate. A nucleic acid target sampleis hybridized with an array of such oligonucleotides and then the amountof target nucleic acids hybridized to each probe in the array isquantified. One preferred quantifying method is to use confocalmicroscope and fluorescent labels. The Affymetrix GeneChip™ Array system(Affymetrix, Santa Clara, Calif.) and the Atlas™ Human cDNA ExpressionArray system are particularly suitable for quantifying thehybridization; however, it will be apparent to those of skill in the artthat any similar systems or other effectively equivalent detectionmethods can also be used. One can use the knowledge of DJ-1 gene todesign arrays of polynucleotides, cDNAs or genomic DNAs for screeningmethods described herein. Such novel pluralities of polynucleotides arecontemplated to be a part of the invention.

In general, typical clinical samples that can be used to determine DJ-1expression include, but are not limited to, blood or blood cells such aswhite blood cells (e.g., granulocytes and monocytes), spinal fluid,synovial fluid, buccal swabs, tissues, urine, saliva, etc.

The expression level of DJ-1 can also be determined by conjugation orligand-binding interaction using a DJ-1 ligand and/or DJ-1 antibody thatis detectably marked. Detectable markers suitable for use in theinvention include any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels in the present invention include biotinfor staining with labeled streptavidin conjugate, magnetic beads (e.g.,Dynabeads™), fluorescent dyes (e.g., fluorescein, texas red, rhodamine,green fluorescent protein, and the like), radiolabels (e.g., ³H, ¹²⁵I,³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horseradish peroxidase, alkalinephosphatase and others commonly used in an ELISA), and colorimetriclabels such as colloidal gold or colored glass or plastic (e.g.,polystyrene, polypropylene, latex, etc.) beads.

The present inventors have found that stimulating a subject toover-express DJ-1 improved a clinical condition associated with aneurodegenerative disease. Without being bound by any theory, it isbelieved that such an improvement is due in some instances at least inpart by tolerance to oxidative stress by selectively upregulating therate limiting step in glutathione synthesis. It was found that when adifferent metabolic insult was imposed, e.g., A53T mutant α-synuclein,it was found that DJ-1 turned on production of the chaperone proteinHsp-70 without affecting glutathione synthesis.

The present inventors have discovered that histone deacetylase (“HDAC”)inhibitors, such as phenylbutyrate, or a derivative thereof, a prodrugthereof, or a salt thereof, increased DJ-1 expression in the N27dopamine cell line and rescued cells from oxidative stress and mutantα-synuclein toxicity. The terms “pro-drug” and “prodrug” are usedinterchangeably herein and refer to a pharmacologically substantiallyinactive derivative of a parent drug molecule that requiresbiotransformation, either spontaneous or enzymatic, within the organismto release the active drug. Prodrugs are variations or derivatives ofthe compounds that have groups cleavable under metabolic conditions.Prodrugs become compounds that are pharmaceutically active in vivo whenthey undergo solvolysis under physiological conditions or undergoenzymatic degradation. Prodrugs may undergo a number ofbiotransformation steps to release the active drug within the organism.Prodrug forms often offer advantages of solubility, tissuecompatibility, or delayed release in the mammalian organism (see,Bundgard, Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985and Silverman, The Organic Chemistry of Drug Design and Drug Action, pp.352-401, Academic Press, San Diego, Calif., 1992). Prodrugs commonlyknown in the art include acid derivatives that are well known to oneskilled in the art, such as, but not limited to, esters prepared byreaction of the parent acids with a suitable alcohol such as diglycerol,triglycerol, etc., or amides prepared by reaction of the parent acidcompound with an amine, or basic groups reacted to form an acylated basederivative. Prodrugs include compounds wherein an amino acid residue, ora polypeptide chain of two or more (e.g., two, three or four) amino acidresidues that are covalently joined through peptide bonds to free amino,hydroxy or carboxylic acid groups of the parent compounds. The aminoacid residues include the 20 naturally occurring amino acids commonlydesignated by three letter symbols and also include, 4-hydroxyproline,hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvalin,beta-alanine, gamma-aminobutyric acid, citrulline homocysteine,homoserine, ornithine and methionine sulfone. Prodrugs also includecompounds wherein carbonates, carbamates, amides and alkyl esters thatare covalently bonded to the above substituents of a compound of theinvention through the carbonyl carbon prodrug sidechain.

Accordingly, some aspects of the invention provide a method for treatinga neurodegenerative disease by administering a histone deacetylaseinhibitor, or a derivative thereof, a prodrug thereof, or a saltthereof. Whether a particular compound is an HDAC inhibitor can bereadily determined, for example, by an in vitro experimentation. Suchexperimental procedures are well known to one skilled in the art.Moreover, many HDAC inhibitors are well known. Exemplary HDAC inhibitorsinclude, but are not limited to, TSA, DPAH, Tubastatin A, MGCD,hydroxamic acids (or hydroxamates), such as trichostatin A, vorinostat(SAHA), belinostat, LAQ824, and panobinostat; cyclic tetrapeptides (suchas trapoxin B), and the depsipeptides; benzamides such as entinostat,CI994, and mocetinostat; electrophilic ketones; and the aliphatic acidcompounds such as phenylbutyrate and valproic acid.

The present inventors have discovered that in mice, phenylbutyratetreatment led to a 260% increase in brain DJ-1 levels and protecteddopamine neurons against 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine(MPTP) toxicity. In a transgenic mouse model of diffuse Lewy bodydisease, long-term administration of phenylbutyrate reduced α-synucleinaggregation in brain and prevented age-related deterioration in motorand cognitive function. Accordingly, the present inventors havediscovered that compounds that upregulate DJ-1 gene expression slow theprogression of a neurodegenerative disease, such as Parkinson's disease,by moderating oxidative stress and protein aggregation.

For the sake of clarity and brevity, the present invention will bedescribed with regard to treating Parkinson's disease using acomposition comprising a compound that increases DJ-1 expression or theactivity of DJ-1 protein. However, it should be appreciated that themethod of the invention can be used to treat other neurodegenerativediseases including, but not limited to, Alzheimer's disease,Huntington's disease, Multiple System Atrophy, Progressive SupranuclearPalsy, Down syndrome, diffuse Lewy body disease, and amyotrophic lateralsclerosis.

Dopamine cell death in Parkinson's disease (PD) results from bothgenetic and environmental factors (1-5). Six genes have been linked toPD including α-synuclein, Parkin, UCHL1, DJ-1, PINK1, and LRRK2 (6-7).α-Synuclein mutations (A53T, A30P, and E46K) cause autosomal dominantforms of PD (8-10). Even in sporadic cases of PD, aggregated α-synucleinhas been found to be a major component of Lewy bodies (11-13). Thetoxicity of mutant forms of α-synuclein results from increased formationof oligomeric and fibrillar aggregates (14-17). The present inventorshave demonstrated, as well as others, that expression of A53T mutantα-synuclein results in protein aggregation and cell death in cultureddopamine neurons (18-22).

Mutations in the DJ-1 gene (PARK7) lead to early-onset, autosomalrecessive Parkinson disease (23-26). Ordinarily, DJ-1 protects cells bya number of mechanisms. The protein can self-oxidize by formingcysteine-sulfinic acid under oxidizing conditions, thereby shifting itspI from 6.1 to 5.8 (27-28). DJ-1 can sequester the cell death proteinDaxx and prevent Daxx-induced apoptosis after oxidative stress (29).DJ-1 can stabilize Nrf2 (nuclear factor erythroid 2-related factor) bypreventing association with its inhibitor protein, Keapl, therebyblocking the subsequent ubiquitination of Nrf2 (30).

The present inventors have shown that overexpression of WT DJ-1 canprotect dopamine neurons from oxidative stress by increasing cellularglutathione (GSH) levels through selective upregulation of the ratelimiting step in GSH synthesis-glutamate cysteine ligase (GCL) (31). Thepresent inventors have also discovered that over-expression of WT DJ-1inhibits A53T human α-synuclein protein aggregation and reduces neuraltoxicity in N27 cells by upregulating heat shock protein 70 (Hsp70)without changing glutathione synthesis. Therefore, it is believed thatDJ-1 acts through independent, distinct mechanisms to protect cells fromdifferent metabolic challenges.

The present inventors have found that over-expression of DJ-1 in thebrain provides broad protection from metabolic insults. DJ-1 isimportant for both the oxidative stress response and the elimination ofabnormal protein aggregates. Recently, histone deacetylase inhibitors(HDACi), such as sodium phenylbutyrate (PB) and sodium butyrate (SB),have shown neuroprotective function in several neurodegenerative diseaseanimal models (32-35). The present inventors have discovered thatphenylbutyrate and sodium butyrate can increase DJ-1 expression andprevent cell death following oxidative stress. In the MPTP mouse model,the present inventors have found that elevating DJ-1 expression by PBadministration reduced the toxicity of MPTP to dopamine neurons. Usingthe newly established Y39C α-synuclein transgenic mouse model ofage-related diffuse Lewy body disease (36), the present inventors haveshown that PB can prevent mutant α-synuclein-induced protein aggregationand improve motor and cognitive function.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting. Inthe Examples, procedures that are constructively reduced to practice aredescribed in the present tense, and procedures that have been carriedout in the laboratory are set forth in the past tense.

EXAMPLES

The abbreviations used are: ALD, adrenoleukodystrophy-related gene; GSH,glutathione; GCL, glutamate cysteine ligase; GFP, green fluorescentprotein; HEK, human embryonic kidney; HRP, horseradish peroxidase; LB,Lewy body; SMN, survival motor neuron; nTg, non-transgenic.

Culture of N27 Cells, HDAC Inhibitor Treatment, and Oxidative StressTreatment:

Dopaminergic cells derived from embryonic day 12 rat mesencephalon andimmortalized with the SV40 large T antigen designated 1RB3AN27, (N27cells) were used (37). N27 cells were cultured in 6-well or 24-wellplates in RPMI 1640 medium containing 10% fetal bovine serum. Cells weretreated with sodium phenylbutyrate and sodium butyrate at differentconcentrations for 48 hr, followed by exposure to varying doses of toxinfor 24 hr.

Culture of N27 Cells, A53T α-Synuclein Adenovirus Transduction, shDJ-1Knockdown of Endogenous DJ-1, and Oxidative Stress Treatment:

N27 cells were cultured in 6-well or 24-well plates in RPMI 1640 mediumcontaining 10% fetal bovine serum and treated with HDAC inhibitor for 48hr. To transduce cells, A53T α-synuclein adenovirus was mixed withculture medium and incubated with cells for 24 hr at a concentration of200 plaque forming units (pfu)/cell. To knock down endogenous DJ-1, N27cells were incubated with adenovirus expressing single hairpin rat DJ-1(Ad-shDJ-1) at 200 pfu/cell as described previously (31). Two days afteradenovirus transduction, cells were exposed to drugs at varying dosesfor 24 hr: H₂O₂ (0-100 μM) and 6-OHDA (0-100 μM).

Immunocytochemistry.

Cultured cells were fixed with 4% paraformaldehyde and processed forimmunocytochemistry as described (31,38). The antibodies included mouseanti-α-synuclein (1:300, Transduction Laboratories); mouse antihumanDJ-1 (1:500, Stressagen); rabbit anti-DJ-1 (1:500, Chemicon); and rabbitanti-TH (tyrosine hydroxylase, 1:200, PelFreez).

MTT Assay and Apoptosis Evaluation.

At the end of each experiment, methylthiazoletetrazolium (MTT) was addedto the culture medium (final concentration 0.4 mg/mL) and incubated fortwo hours. Cell viability was measured by a microplate reader asdescribed (31,38). The nuclear dye Hoechst 33258 (10 μg/ml) was used tovisualize and count apoptotic cells.

Western Blotting.

N27 cells were cultured in 6-well plates and treated with compounds asdescribed above. Cells were lysed in a dissociation buffer containing 50mM Tris-HCl, 10 mM NaCl, 0.1% Triton X-100 plus protease inhibitorcocktail (Roche). The mouse brain tissues were dissected and quicklyfrozen in dry ice. Tissues were thawed on ice and homogenized indissociation buffer with protease inhibitors (Roche). Proteinconcentration was determined by the BCA method (Pierce). Fifty μg ofprotein was separated on 10% SDSPAGE gel and transferred to anitrocellulose membrane. After blocking nonspecific binding, membraneswere incubated with antibodies to DJ-1 (1:5000, Chemicon), α-synuclein(1:3000, Transduction Laboratories), TH (1:2000, PelFreez), and β-actin(1:4000, Sigma). Blots were incubated with HRP-conjugated secondaryantibody (1:10,000; Jackson Immuno Research), followed bychemiluminescent detection (Perkin Elmer Life Sciences) (31,38).

Luciferase Assay.

The luciferase assay was based on the pGLuc-Basic vector (NEB). Thehuman DJ-1 2 kb promoter area was amplified by PCR using genomic DNAfrom HEK293 cells. The human DJ-1 promoter was then cloned into thepGLuc-Basic vector, and that reporter vector was transfected into HEK293cells. G418 (200 μg/ml) was applied to the cultures, and resistantclones were selected and assayed for luciferase activity. Stable celllines expressing pDJ1-Luciferase were treated with PB and SB for 48 hrat various concentrations in 24-well plates. Culture medium samples (20μl) were incubated with Gaussia luciferase assay substrate (NEB), andluminescence was measured in a 96-well plate reader (BioTek Synergy HTMulti-Mode microplate reader).

Phenylbutyrate and Butyrate Treatment in Mice.

Adult C57BL/6 mice (4-6 months old) were treated with PB and SB indrinking water for 14 days. The PB and SB were dissolved in water atconcentrations of 500, 1000, 1500, and 2000 mg/L. Mice typically drink4-5 ml of water per day, and their drinking volumes were not affected bythe addition of PB or SB. Control animals received water with sodiumchloride added to the same molarity as the sodium in the drug-treatedanimals. Brain tissues were dissected and immediately frozen in dry icefor Western blot analysis using DJ-1, α-synuclein and β-actinantibodies.

MPTP Injection into Mice.

Adult C57BL/6 mice (4-6 months old) were treated with PB in drinkingwater (1000 mg/L, 5.4 mM) for two weeks, followed by injection of1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP-HCl, dissolved insterile saline, Sigma-Aldrich) four times at 2-hr intervals (20 mg/kg,i.p.). Treatment with PB continued after MPTP injection. Control animalsreceived identical injections of MPTP, but had only sodium chlorideadded to drinking water as described above. One week after MPTPlesioning, mouse brain tissues were dissected for Western blot and HPLCanalysis.

HPLC Analysis of Dopamine and DOPAC.

Mouse striatum was frozen in dry ice, sonicated in ice-cold 0.2 mperchloric acid and centrifuged at 15,000 g for 15 min at 4° C. Analiquot (5 μl) of the supernatant solution was analyzed by HPLC equippedwith an electrochemical detector (CoulArray system ESA Model 5600; ESA,Boston, Mass., USA), a pump (ESA Model 580) set at 1.5 mL/min, and areverse-phase C18 column (3 μm, 100 A˜4.6 mm, Waters, Milford, Mass.,USA). The mobile phase was composed of 100 mm citric acid, 2% methanol,1 mm EDTA and 5 mg/L sodium octyl sulfate (pH 3.0).

Transgenic Mice Expressing Human Y39C α-Synuclein Under Mouse Thy-1Promoter.

Y39C human α-synuclein transgenic mouse model has been described by thepresent inventors (36). Briefly, human Y39C α-synuclein cDNA was clonedinto the mouse pThy-1 vector at the Not1 site. The construct wasmicroinjected into mouse oocytes, and founder mice were identified byPCR and Southern blotting analysis. Mice were bred to establish stabletransgenic lines. Expression of human Y39C α-synuclein in thesetransgenic mice was determined by immunostaining and Western blottingwith antibodies specific to human α-synuclein (LB509).

Drug Treatment in 1-Synuclein Transgenic Mice.

The Y39C α-synuclein transgenic mice were divided into younger (6-8months=Young Tg) and older (10-12 months=Old Tg) groups. Each groupreceived PB (1000 mg/L, 5.4 mM) or vehicle (NaCl) in the drinking waterfor 3 months. Animals were tested for rotarod and water maze performanceafter 6-weeks and 12-weeks of treatment.

Rotarod Test.

Mice were tested for their ability to run on a 3 cm diameter rotatingrod (rotarod) at speeds ranging from 3 to 33 rpm. The protocol consistedof two phases: habituation (Day 1) and rotarod training/testing (Days 2to 5). During habituation on Day 1, the mice were trained to remain onthe rotarod at 3 rpm. During training/testing on Days 2 to 5, mice wereplaced on the rotating rod at a constant speed for three one-minutetrials with a 5 minute rest interval between trials. Each test day, thespeed was increased, reaching 33 rpm by Day 5. The time the mice spenton the rotarod without falling was recorded for each trial.

Morris Water Maze Testing.

Spatial learning was assessed using the Morris water maze. The mazeincluded a circular tank (120 cm in diameter) filled to 10 cm below theedge of the tank with 27° C. water that was made opaque by the additionof non-toxic black ink. A circular escape platform (10 cm in diameter)was located 1 cm below the surface of the water in a constant locationin the northwest quadrant of the tank. Mice were first acclimated to themaze during three trial habituation sessions. Each testing sessionconsisted of three consecutive days with four trials per day. Theplatform was invisible in the pool, and mice were allowed to swim for 60sec before being returned to the home cage. The latency from alltraining and testing sessions was collected.

Immunohistochemistry and α-Synuclein Staining.

The mice were sacrificed by deep anesthesia followed by intracardiacperfusion with saline and 4% paraformaldehyde. The mouse brains werecryosectioned at 40 μm. Immunohistochemistry was performed usingantibodies to human α-synuclein (LB509, 1:500). Immunostaining wasdeveloped with diaminobenzidine (DAB). Tissue sections were examined forthe Lewy body-like inclusions.

Statistics.

All experiments were repeated at least three times. Data were analyzedusing multivariate ANOVA and the Fisher LSD post hoc test. Significancewas set at p<0.05. Values are shown as mean±SEM.

Results

Sodium Phenylbutyrate (PB) and Sodium Butyrate (SB) Increase DJ-1Expression in N27 Cells and HEK293 Cells.

Using the N27 rat dopaminergic cell line, a number of compounds werescreened for their ability to increase DJ-1 expression. From thisscreen, it was found that sodium phenylbutyrate and sodium butyrateincreased DJ-1 protein levels to 300% of control after two daystreatment as shown in FIG. 1A. Both compounds had peak effects atconcentrations of 0.15-0.3 mM (**p<0.01 compared to control, FIG. 1B-C).To test if PB and SB directly upregulated DJ-1 gene transcription, anHEK293 reporter cell line was created which stably expressed the humanDJ-1 promoter-luciferase construct. The HEK293 reporter cells weretreated with PB and SB for 48 hr, followed by luciferase assay. It wasfound that both PB and SB increased luciferase activity as well as DJ-1protein levels (*p<0.05, **p<0.01 compared to control, FIG. 1D-E). Theseresults indicate that enhanced DJ-1 gene transcription is responsiblefor the higher DJ-1 protein levels seen after PB and SB treatments.

Sodium Phenylbutyrate Protects N27 Cells from Oxidative Stress andα-Synuclein Induced Toxicity.

The present inventors have previously shown that overexpression of theDJ-1 gene through adenoviral transduction can make N27 cells moreresistant to oxidative stress and mutant α-synuclein toxicity (31). Tosee if phenylbutyrate could replicate these protective effects, N27cells were treated with 0.15 mM sodium phenylbutyrate for two days andthen subjected cells to oxidative stress for 24 hr. FIGS. 2A and B showsample images of N27 cells with or without PB followed by H₂O₂treatment, in which cells were identified by GFP adenovirus expression.Quantitative results showed that PB treatment significantly increasedcell viability after exposure to hydrogen peroxide (H₂O₂) and6-hydroxydopamine (6-OHDA) compared to controls (*p<0.05, **p<0.01,FIGS. 2E,F).

The present inventors have earlier demonstrated that expression of A53Tmutant human α-synuclein in N27 cells led to cell death withα-synuclein-positive cytoplasmic aggregates (31,38). In the presentexperiments, N27 cells were treated with PB for two days. Cells werethen exposed to adenovirus expressing A53T mutant α-synuclein foranother two days. FIGS. 2C and 2D show sample images of N27 cells withor without PB treatment followed by A53T α-synuclein expression, inwhich cells were identified by α-synuclein immunostaining. It was foundthat PB treatment increased cell viability and reduced the number ofcells with α-synuclein aggregates (*p<0.05, **p<0.01 compared tocontrol, FIG. 2G-H). These data indicate that phenylbutyrate can protectdopamine cells from oxidative stress and mutant α-synuclein toxicity.

Knocking Down DJ-1 Blocks Phenylbutyrate Effects in N27 Cells.

To test whether DJ-1 is needed for the protective actions of PB, thepresent inventors have used knock-down technology. With an adenovirusexpressing shDJ-1 (31), DJ-1 gene expression was knocked down in N27cells (FIG. 3A) and then tested whether PB could rescue cell deathresulting from H₂O₂ (75 μM or 100 μM) and 6-OHDA (60 μM or 80 μM).Results showed that DJ-1 knockdown in N27 cells effectively abolishedthe neuroprotective effect of PB against H₂O₂ and 6-OHDA-inducedtoxicity (compare columns with horizontal and vertical bars to controlin FIGS. 3B-C), while naive N27 cells with PB treatment significantlyimproved cell viability (*p<0.05, **p<0.01 compared to control, FIGS.3B, 3C).

These in vitro experiments demonstrate that phenylbutyrate can turn onexpression of the DJ-1 gene and protect N27 cells from oxidative stressand mutant α-synuclein toxicity. Importantly, activation of DJ-1 isrequired for the phenylbutyrate effect.

Sodium Phenylbutyrate and Sodium Butyrate Increase DJ-1 Expression inMice.

With the finding that PB and SB provide a way to turn on DJ-1 geneexpression in vivo and thereby protect brain from neurotoxic stress,mice were treated with these drugs. Because phenylbutyrate is stable insolution and has a very short half-life (50 min) in vivo, drugs weredelivered in drinking water. Adult C57BL/6 mice were treated with PB orSB at various concentrations in drinking water for two weeks. Controlmice received water with equimolar concentrations of sodium chloride. Itwas found that all treatment and control groups consumed similar volumesof water, and the intake for all was in the range expected for normaldaily fluid consumption (data not shown). Animals were sacrificed by anoverdose of anesthetic, and brains removed and frozen on dry ice. Braintissues were analyzed for DJ-1 protein levels by Western blotting.Results showed PB and SB significantly increased DJ-1 levels (*p<0.05,**p<0.01 compared to control, FIGS. 4A-C).

To test whether the increase in DJ-1 was caused by a global increase ingene and protein expression, endogenous α-synuclein protein levels werealso measured as shown in FIGS. 4A-C. α-Synuclein concentrations werenot changed after drug treatment, suggesting that PB and SB showedselectivity in producing an increase in DJ-1 expression in brain. Tostudy the time course of DJ-1 expression during chronic drugadministration, DJ-1 levels were examined in mice treated with PB in thedrinking water for 1, 2, and 3 months. It was found that brain DJ-1reached plateau values after two weeks of PB, and those levels weresustained for 1 to 3 months of treatment (data not shown). These dataindicate that PB can provide sustained elevation of DJ-1 expression inmouse brain.

Sodium Phenylbutyrate Protects Dopamine Neurons from MPTP-InducedNeurotoxicity.

To evaluate whether pre-treatment with PB can protect dopamine neuronsin the MPTP mouse model of Parkinson's disease, adult C57BL/6 mice weretreated with PB in the drinking water (1000 mg/L) for two weeks. Theywere then injected with MPTP 4 times at 2 hr intervals (20 mg/kg, i.p.,each injection). One week after MPTP injections, mice were sacrificedfor biochemical and histological analysis. It was found thatPB-pretreatment significantly increased striatal dopamine levels asmeasured by HPLC (*p<0.05 compared to MPTP alone, FIG. 5A). PB treatmentalso increased tyrosine hydroxylase (TH) protein levels in striatum(Western blot, **p<0.01 compared to MPTP alone, FIGS. 5B-5C).Furthermore, DJ-1 levels in striatum were significantly increased inPB-treated mice (Western blot, **p<0.01 compared to MPTP alone, FIGS.5B, 5D). It was also found that PB treated mice had significantly highernumbers of TH positive dopamine neurons in the substantia nigra (*p<0.05compared to MPTP alone, FIG. 5E). Sample immunohistochemical imagesshowing increased survival of TH-positive dopamine neurons in substantianigra are presented in FIGS. 5F-H. These data indicate thatphenylbutyrate can protect dopamine neurons from MPTP neurotoxicity.

Sodium Phenylbutyrate Prevents Age-Related Motor and Cognitive Declinein Mice with Diffuse Lewy Body Disease.

The present inventors have created a transgenic mouse model expressing atyrosine-to-cysteine (Y39C) mutant human α-synuclein (36). Because thetransgene is expressed under control of the Thy 1 promoter, mutantprotein accumulates throughout the brain in all neurons. These animalshave progressive, age-related decline in motor and cognitive function.Histopathology shows Lewy body-like α-synuclein inclusions in neurons.The age-related behavioral and neuropathologic phenotypes havesimilarities to PD and diffuse Lewy body disease.

To test whether PB treatment can prevent the development of behavioraland neuropathological deficits in this transgenic mouse model,transgenic mice were divided into a Young Tg group (age 6-8 months) andan Old Tg group (age 10-12 months). Mice were treated with PB (1000mg/L, 5.4 mM) or vehicle (NaCl, 310 mg/L) in drinking water for threemonths. Mice were tested for motor function after 6 weeks and 12 weeksof treatment using a rotarod with increasing speed β-33 RPM). Resultsshowed that PB treatment did not change motor function in Young Tg miceat 6-weeks or 12-weeks of therapy when animals were up to 11 months ofage (FIGS. 6A, 6C). By contrast, in Old Tg mice, motor functiondeteriorated progressively with age in animals that were 13 to 15 monthsold at the end of the study. PB treatment prevented the decline andsignificantly improved motor function in Old Tg mice at both 6-week and12-week tests (*p<0.05, **p<0.01 compared to vehicle, FIGS. 6B, 6D). TheOld Tg mice treated with PB performed similarly to Young Tg mice(compare FIGS. 6C and 6D).

Mice were tested for cognitive function at 6 weeks and 12 weeks oftreatments using a Morris water maze. In these transgenic mice, thepresent inventors have reported that water maze performance deterioratesas mice age (36). In the current studies, it was found that Young Tgmice performed well in the Morris water maze, and PB treatment did notchange water maze performance at 6-week and 12-week tests (FIGS. 7A,7C). In Old Tg mice, PB treatment prevented the age-related decline inwater maze function at both 6-week and 12-week tests (*p<0.05, **p<0.01compared to vehicle, FIGS. 7B, 7D). These results show that sodiumphenylbutyrate can improve motor and cognitive function in agedtransgenic mice.

Sodium Phenylbutyrate Reduces α-Synuclein Aggregation and IncreasesExpression of DJ-1 in Old Transgenic Mouse Brain.

α-Synuclein aggregation was examined in transgenic mice treated with PB.Brain tissue lysates were separated in SDS-PAGE and probed withα-synuclein antibody. FIG. 8A shows Western blot images from young andold mice. Brain from a 15-month old transgenic mouse (15 m Tg-PB) showedintense α-synuclein oligomer fractions in cortex, striatum andhippocampus. PB treatment dramatically reduced oligomer formation in anage-matched transgenic mouse (15 m Tg+PB) (FIG. 8A). In 10-month oldYoung transgenic animals, oligomer accumulation had not yet occurred,and the effect of PB was minimal (10 m Tg-PB and +PB) (FIG. 8A). FIG. 8Bpresents the ratios of α-synuclein oligomer to monomer. The very highlevel of oligomer in old transgenic mice (Old w/o PB) is dramaticallyreduced by treatment (**p<0.01, FIG. 8B). In these same treatmentgroups, it was found that brain DJ-1 protein levels were significantlyincreased in both young and old transgenic mice treated with PB comparedto age-matched mice without PB treatment (*p<0.05, FIGS. 8C-D).

α-Synuclein immunostaining was performed in mouse brain sections usingLB509 antibody. The PB treatment had little effect on the number ofneurons with Lewy body-like inclusions in young mice (sample images from10 m Tg mice with and without PB are shown in FIGS. 8E, 8F). However, inold mice, the PB treatment greatly reduced the number of neurons withLewy body-like inclusions (sample images from 10 m Tg mice with andwithout PB are shown in FIGS. 8G, 8H). Control staining from 15 monthold non-Tg mice is shown in FIG. 81. The percentage of α-synucleinpositive neurons with Lewy body-like inclusions is shown in FIG. 8J(**p<0.01, PB compared to Vehicle). These data indicate thatphenylbutyrate can increase DJ-1 expression, reduce α-synuclein oligomerformation, and prevent age-related decline in motor and cognitivefunction in a transgenic mouse model of diffuse Lewy body disease.

Glycerol Phenylbutyrate.

Glycerol phenylbutyrate (i.e., phenylbutyrate triglyceride) exhibitedmotor performance improvement in transgenic mice overexpressing humanα-synuclein in brain neurons. Glycerol phenylbutyrate is a liquidpro-drug manufactured by Hyperion Pharmaceuticals and is also namedH-100. The drug is shown to be metabolized in the gut to produce theactive compound phenylbutyrate.

Transgenic mice were genetically programmed from birth to develop a“Parkinson-Plus” condition by depositing an abnormal form of a proteincalled Y39C-α-synuclein in neurons of the brain under control of theThy1 promoter (see Zhou et al., J. Biol. Chem., 2008, 283, 9863-70). Atone year of age, 8 mice were treated with daily glycerol phenylbutryrate(solid line) and another 8 mice were treated with the inactive compoundglycerol (dashed line). After 6 weeks of daily treatment, animals wereplaced on a rotating rod (Rotarod) at different speeds for up to 5minutes. Animals which could not keep up with the turning rod fell off.The time they were able to stay on the rod was noted. As shown in FIG.9, transgenic animals treated with daily glycerol phenylbutyrate wereable to stay on the rod longer than animals receiving only glycerol.These differences were statistically significant at the higher speeds,21 and 26 rpm. At the highest speed, 33 rpm, all animals fell off therod after about 50 seconds. The improved motor performance is similar tothat observed in an earlier study of the same strain of transgenic micereceiving sodium phenylbutyrate in their drinking water (Zhou et al, J.Biol Chem., 2011, 286: 14941-51).

Phenylbutyrate Works by Turning on the Protective Gene DJ-1 in BrainCells.

DJ-1 makes cells resistant to oxidative stress and resistant toaccumulation of abnormal proteins by promoting production of exosomes.

Phenylbutyrate upregulates the protective gene DJ-1 with a 50-foldincrease in mRNA in N27 dopamine neurons in tissue culture. Exposure ofcells to hydrogen peroxide also increases DJ-1 mRNA. As shown in FIG.10, in cells pretreated with phenylbutyrate for 48 hours, thecombination of phenylbutyrate and H₂O₂ led to a greater increase in DJ-1mRNA (256-fold). Cell stress created by the overexpression of A53Tmutant human alpha-synuclein (A53T) increased DJ-1 gene expression,while the combination of phenylbutyrate and A53T caused an even greaterincrease. Not shown is the fact that cells treated with phenylbutyratehave substantially better cell survival than those exposed to H₂O₂ andA53T without phenylbutyrate. When DJ-1 gene expression was blocked byanti-sense RNA, the protective effects of phenylbutyate were eliminated(Zhou et al, J. Biol Chem., 2011, 286: 14941-51).

As shown in FIG. 11, the oxidative stress pathway Nrf2 was upregulatedby phenylbutyrate with a 50-fold increase in mRNA. Interestingly,overexpression of the mutant protein A53T did not turn on the Nrf2 gene.

In addition, the lysosomal pathway Lamp2 was activated by phenylbutyratein N27 dopamine neurons. See FIG. 12. The drug increased expression byabout 30-fold while H₂O₂ led only to a 4-fold increase. Pretreatmentwith phenylbutyrate for 48 hr followed by H₂O₂ increased expression by200-fold. Similarly, A53T increased expression of Lamp2 as part of thecells' response to abnormal protein stress. Phenylbutyrate amplifiedthat response to a 400-fold increase in gene expression.

FIG. 13 shows the bar graph of tissue culture results in N27 dopamineneurons showing that the vesicle-exosome protein Vamp2 was upregulatedparticularly by mutant protein expression with the addition ofphenylbutyrate. As can be seen, the exosome marker Vamp2 was upregulatedparticularly by overexpression of mutant protein A53T human α-synuclein(80-fold). The addition of phenylbutryrate increased that overexpressionto 200-fold.

Discussion

The present disclosure describes the neuroprotective effects of HDACinhibitors (e.g., phenylbutyrate) in both cell culture and in mousemodels. It was found that phenylbutyrate can upregulate DJ-1 mRNA andprotein levels in rat dopaminergic N27 cells and HEK293 cells. Increasedexpression of DJ-1 renders cells more resistant to oxidative stress andα-synuclein-induced toxicity. Blocking DJ-1 activation withanti-sense-DJ-1 interferes with the physiologic protection. In mice, thepresent inventors have discovered that phenylbutyrate given throughdrinking water can increase brain DJ-1 levels. Upregulation of DJ-1resulted in neuroprotection for dopamine neurons against MPTP toxicity.Moreover, phenylbutyrate given to transgenic mice that overexpress amutant form of α-synuclein prevented oligomer formation in brain andstopped the age-related decline in motor and cognitive function.

Phenylbutyrate is a histone deacetylase inhibitor (HDACi) and has beenshown to be neuroprotective in animal models of Huntington's disease,spinal muscular atrophy, and amyotrophic lateral sclerosis(33-34,39-43). The drug has been shown to protect dopamine neurons fromdeath in the MPTP mouse model of Parkinson's disease (35).Phenylbutyrate can protect dopamine neurons from rotenone-induced celldeath. In transgenic mice expressing both A53T and A30P humanα-synuclein, treatment with phenylbutyrate can improve behavioralfunction and reduce neuropathology (44-45). These prior studies did notpropose a link between phenylbutyrate and the activation of DJ-1 geneexpression.

As a histone deacetylase inhibitor, phenylbutyrate can increaseacetylation levels of histone H3 and H4, thereby promotingtranscriptional activation (33,46). Previous studies have demonstratedthat phenylbutyrate can increase expression of many genes includingantiapoptotic genes, components of ubiquitinproteosomal pathways,nuclear factor NF-kB p50, survival motor neuron 1 (SMN1), andadrenoleukodystrophy-related gene (ALD) (40-41,46-47). Other reportshave shown that histone deacetylase inhibitors can activate INK4d andDRS genes through the Spl binding site in the promoters (48-50). Becausethe DJ-1 gene promoter contains Spl binding sequences (51), it ispossible that phenylbutyrate increases DJ-1 gene expression by increasedbinding of Spl to the DJ-1 promoter. Our DJ-1 promoter-luciferasereporter assay in HEK 293 cells has provided additional evidence thatphenylbutyrate can increase DJ-1 gene expression. The present inventorshave found that blocking DJ-1 expression with shDJ-1 blocks theneuroprotective effects of phenylbutyrate.

Increased DJ-1 levels protect against oxidative stress and otherbiochemical toxicity through multiple pathways. DJ-1 can stabilize Nrf2,a master regulator of antioxidant transcriptional responses, by blockingassociation with its inhibitor protein Keapl, thereby preventingubiquitination of Nrf2 (30). DJ-1 can also sequester the cell deathprotein Daxx in the nucleus and prevent Daxx-induced apoptosis afteroxidative stress (29). Recent reports show that DJ-1 can work inparallel with the PINK1/parkin pathway to maintain mitochondrialfunction in the presence of an oxidative environment (52-53). Inaddition, DJ-1 can act as a redox-dependent molecular chaperone toinhibit α-synuclein aggregate formation (54-55). The present inventorshave shown that DJ-1 can increase glutathione synthesis after oxidativestress and can upregulate heat shock protein 70 (Hsp70) to blockα-synuclein aggregation (31). While the majority of PD patients do notcarry DJ-1 gene mutations, results disclosed herein indicate thatincreasing DJ-1 expression to supra-normal levels can make dopamineneurons resistant to neurotoxic insults. Drugs that enhance DJ-1 geneexpression may be neuroprotective for all Parkinson's disease patients.

Phenylbutyrate has additional metabolic effects. It can be a chaperonemolecule. As a chemical chaperone, phenylbutyrate can bind and masksurface-exposed hydrophobic segments of unfolded proteins and therebystabilize protein structure in the native conformation, reducingendoplasmic reticulum (ER) stress (56-58). In α-synuclein transgenicmouse model, it is possible that phenylbutyrate directly stabilizesmutant α-synuclein and prevents the formation of high molecular weightoligomers and fibrils.

As disclosed herein, the present inventors have found that increasedexpression of DJ-1 prevents progression of motor and cognitivecomplications in a transgenic mouse model of diffuse Lewy body disease.Accordingly, various neurodegenerative diseases can be treated byadministering a compound that increases expression of DJ-1. For example,phenylbutyrate can increase DJ-1 expression and prevent progression ofdisease in patients with idiopathic Parkinson's or diffuse Lewy bodydisease.

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The foregoing discussion of the invention has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the invention to the form or forms disclosed herein. Althoughthe description of the invention has included description of one or moreembodiments and certain variations and modifications, other variationsand modifications are within the scope of the invention, e.g., as may bewithin the skill and knowledge of those in the art, after understandingthe present disclosure. It is intended to obtain rights which includealternative embodiments to the extent permitted, including alternate,interchangeable and/or equivalent structures, functions, ranges or stepsto those claimed, whether or not such alternate, interchangeable and/orequivalent structures, functions, ranges or steps are disclosed herein,and without intending to publicly dedicate any patentable subjectmatter.

What is claimed is:
 1. A method for treating a neurodegenerative diseasein a subject, the method comprising administering to the subject atherapeutically effective amount of a compound that increases DJ-1 geneexpression or DJ-1 protein activity.
 2. The method of claim 1, whereinthe compound increases DJ-1 protein expression by at least 50%.
 3. Themethod of claim 1, wherein the compound increases DJ-1 proteinexpression by at least 100%.
 4. The method of claim 1, wherein thecompound is administered to the subject no more than 10 units per day.5. The method of claim 1, wherein the neurodegenerative diseasecomprises at least one selected from the group consisting of Parkinson'sdisease, Alzheimer's disease, Huntington's disease, Multiple SystemAtrophy, Progressive Supranuclear Palsy, Down syndrome, diffuse Lewybody disease, and amyotrophic lateral sclerosis.
 6. The method of claim1, wherein the compound is a histone deacetylase (HDAC) inhibitor. 7.The method of claim 1, wherein the compound upregulates DJ-1 geneexpression.
 8. The method of claim 1, wherein the compound increasesDJ-1 protein activity.
 9. The method of claim 1, wherein the compoundcomprises phenylbutyric acid, butyric acid, a salt thereof, a prodrugthereof, or a derivative thereof.
 10. The method of claim 1, wherein thecompound comprises phenylbutyrate, or a prodrug thereof, or a derivativethereof.
 11. The method of claim 1, wherein the composition comprisesphenylbutyrate glycerol.
 12. A method for treating a neurodegenerativedisease in a subject, the method comprising administering to the subjecta therapeutically effective amount of a compound that increases DJ-1expression level by at least 150%, wherein the administering increasesDJ-1 expression in the subject, thereby reducing amounts of neuron deathin the subject.
 13. A method for protecting dopamine neurons from aneurotoxic insult in a subject, the method comprising administering tothe subject a compound that increases DJ-1 expression in dopamineneurons, thereby increasing dopamine neuron survivability in thesubject.